SPECIFIC DOMAINS OF LNK (SH2B3) BIND AND REGULATE TPOR STABILITY AND SIGNALLING

The mechanism of thrombopoietin receptor (TPOR)/JAK/STAT signalling regulation by the negative regulator, LNK (an SH2-containing adaptor protein) is still not fully understood. Improved understanding of these mechanisms will reveal novel areas for drug development to improve current treatment strategies for haematological malignancies such as myeloproliferative neoplasms and leukaemia as mutations in LNK has been shown to be involved in these diseases. Domains of LNK that may interact with and regulate JAK2 and/or TPOR activity in co-operation with its SH2 domain were investigated. The SH2 domain of LNK and its family members has been previously shown to bind to JAK2 (phosphorylated Tyr813). Through our signalling assay, we found that the proline dimerisation domain (Pro/DD), besides the SH2 domain, is crucial to maintain LNK inhibitory effects and that deletion of this domain enhanced JAK2-STAT3 and ERK1/2 signalling. Our co-immunoprecipitation results also suggested that the Pro/DD is a potential site that associates with TPOR whilst its SH2 anchor to JAK2. Additionally, we showed that LNK preferentially binds TPOR when JAK2 is present (the presence of JAK2 aids in stabilising this interaction) and that LNK plays a role in maintaining TPOR stability. Overall, we discovered a novel site of LNK, the Pro/DD, which plays a role in inhibiting the JAK-STAT pathway via TPOR. This discovery will help us to better understand how LNK inhibits this pathway and in turn will aid in the development of LNK mimetics.